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bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation

Vision

Until today, 13.628 biobrick sequences[1] have been submitted to partsregistry, thereof 102 reporter units, 12 signaling bricks and xx sensing parts. Since there, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.[2][3][4]

The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting any arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.

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Generally speaking, the above descirbed adapter has to meet the following requirements:

  • Universality
The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.
  • Scalability
Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.
  • Biological orthogonality
Interfering with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.
  • Logic
The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).


Several biological logic units, devices and circuits have been developed so far[5], but to our knowledge, none was shown to meet all requirement listed above.

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Implementation

To functionally connect BioBricks there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.

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How to connect BioBricks

Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practise and consists of four major elements:

Examples of a molecular network. Molecules are indicated as lines, whereas black represents input molecules, red indicates transmitter molecules, switches are green and labeled with their type and output molecules are drawn blue.
  • Input elements
  • Transmitter molecules
  • Logic gates
  • Output elements

These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a transmitter molecule that is unique for this input molecule. All transmitters belong to the same type of molecule and share a common design. However each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize and bind the corresponding transmitter molecules. Depending on the type of the logic gate (AND, OR or NOT[6]), an output molecule is only created if both transmitter molecules are present (AND), at least one of two transmitter molecules is present (OR) or if no transmitter is present at all (NOT). Consequently a certain subset of output molecules are generated according to the presence of transmitter molecules, thus also according to the corresponding BioBricks.

Computer vs. molecular network
Logic gates in a molecular network can be compared to transistors used in a computer where billions of transistors are incorporated. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way of being connected by wires in a special sequence allows specifically adressing only subset of other transistors by an input signal. However, spatial connections are not possible in a living cell. The wiring within a cell relies on the specific interaction between the transmitter molecule and the corresponding logic gates. The main difference is, that each logic unit posses a different xxx . Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially is limited to small circuits so far. Our project aims to establish a real molecular transistor. Therefore, our smallest switching unit consist of two subunits, a transistor site and a recognition site,which equals Nevertheless,
Again, these output molecules are transmitter molecules and can in turn interact with another subset ("layer") of logic gates. In theory many layers of logic gates can be connected by transmitters. The last layer of logic gates has to generate transmitter molecules, that do not interact with logic gates but induce output molecules, such as Biobricks, instead.

Summarizing, the network established a connection between input BioBrick and output BioBrick in a functional manner.
Having addressed the basic layout of the molecular network, the next step is to decide what type of molecules can perform the required functions. For our project we used RNA for transmitter molecules as well as for logic gates. Several advantages result from the utilization of RNA as the central element:

  • During the last years, many Biobricks were designed that are sensivtive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently is capable to produce a specific transmitter RNA molecule. These BioBricks can therefore be used as input molecules just be exploiting their transcription functionality.
  • The use of RNA also allows the integration of various output proteins including all BioBricks. Since all logic gates produce RNA, they can also produce functional mRNA encoding any protein.
  • If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact with each other. Furthermore, a library of interaction transmitters and gates can be generated in silico.
  • RNA production is fast and energy saving for a cell containing the adapter.
  • As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.

Design and functional principle of logic gates

The concept introduced above provides a framework that can potentialy serve as an universal adapter between different BioBricks. However, the logic gates have not been specified more precisely so far. Generally speaking, the logic gates have to posses the following characteristics:

  • Logic gates, such as AND, OR and NOT, have to be implemented.
  • All logic gates have to recognize the corresponding transmitter RNA and, in responds, produce an output transmitter molecule.
  • Logic gates should follow a basic design rule, in such a way, that their creation can be automated in silico.
  • The responds function of all logic gates should be comparable to each other.

In order to build a logic gates for our bioLOGICS system we will first create a simple toggle switch. A toggle switch can be activated by one RNA and produces an RNA output. In contrast to a logic gate, a toggle switch does not perform logic operations. However by combining two toggle switches, logic gates can be created. The following text will first describe how a toggle switch works and secondly, will demonstrate how logic gates such as AND and OR can be created by combining two toggle switches.

Toogle Switch

  • switching unit

The switching unit is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. The principle idea of our switching unit relies on such systems occurring in nature.
The sticking point of our switches is the fact that they all consist of THE SAME switching unit, id est having found one functional "switchable" terminator will allow almost unlimited upscaling. This is the main difference to previous works on this field, which always required developing a new shifting principle for each switch.[2][2][2] Beside the extendability, this principle provides a comparable on/off shifting rate, which avoids complex concentration depended fine tuning of interacting molecular circuits. MÖGLICHST KLAREN BANDPASS --> ja nein Sache. Dazu krasse termintoren mit enhancing der outputs. Additionally, this should allow to set up a binary system within cells, to a certain extend, nota bene. This makes our switches more similar to transistors than ...

  • recognition site

It defines the specific access of one of our toogle switches by an input molecule. Therefore, a unique recognition element is assigned to each switch. This allows to arrange and interconnect numerous of these switches in a specif logical order, comparable to wires connecting different transistors.
It is implemented by simply putting a random sequence with arbitrary length (has to be optimized) in front of the switching unit.

signals, the inputs, transmitters and outputs of the bioLOGICS toggle switches

Signals present the "trigger" to shift switches between the switches´ on and off-state. It requires the ability to change the terminators secondary structure and cause antitermination, BUT only if a special recognition site is detected. Thus, each signal consists of a trigger unit, interacting with the switches´ switching unit and a specifity site, interacting with the terminators recognition site. Practically, the shift between the two terminator states is induced by a complementary RNA-sequence, influencing the terminators secondary structure. But in contrast to previous work [2], we designed this synthetic trigger unit in such a manner it is not able to change the terminator´s state on its own, but only in combination with the specificity site, which is complementary to the recognition site.

The challenge is to arrange and optimize these elementary building blocks, that a trigger unit is only able to switch in combination with its respective specificity site. This was done by NUPACK simulations, presented in section in silico design .

Putting it all together: the switching process


nur vorläufiges bildchen!
Figure xxx illustrates the functional principle of the designed switch. The switch is implemented in the front a desired output. So in the ground state, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain, which then causes the RNA polymerase to stop transcription and fall off the DNA and no output RNA will be produced.

On the other hand, in the presence of a signal, this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids transcription termination. In detail, the specificity unit binds the recognition unit and serves as toehold, which will thermodynamically allow the trigger unit to perform a strand displacement and open the stem loop structure, allowing the polymerase to read through and form the output RNA.
Summing up, we use can use this concept to create a switch that can be toggled by a transmitter RNA molecule and in responds, produces another transmitter RNA.

Challenges are

  • to find a suitable terminator construct and design a complementary trigger unit which is only functional in combination with a specificity site - meaning an optimization of the thermodynamically parameters (seein silico design)
  • to investigate whether the signal/switch interaction reaction is really on a timescale

to be competitive to terminator formation - meaning an comparision of kinetic parameters (see Modeling page)

The bioLOGIC toggle switch with regard of logical operations

As described, each switch can be accessed by a specific RNA-signal molecule, illustrating the input. In turn, another RNA-signal molecule will be produced if the switch shifts its state, now being the output of one switch and at the same time, a possible input for the following switch or several ones. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.

To easen the building of logical networks, applying mathematical logcis, e.g. Boolean logics like in computaltion science would be worthwile. It is possible to establish general Boolean operators with our switches and thus build "logical modules".


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Since AND/OR/NOT are the most easy logic operations modularisable with our switches and can substitute all remaining operations, we exemplarly designed them.

  • AND consists of a parallel circuit of two switches

AND.png
  • OR is implemented by connecting two switches in series
    OR.png
  • NOT contains its respective signal molecule intrinsic, so via intramolecular interaction, the antitermination is the initial state. The signal is composed of the same components as usual but its sequence is complementary.
    NOT.png

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-->wohin damit??? der schöne text von wuschelhaissi: The initial signal can be provided by various metabolic compounds and stimuli, by simply using one of the many sensory systems or inducable promoters submitted to the Partsregistry in the last years. All those parts can now be serve as inputs for our network. BioLOGICS provides a new way to use the whole potential of iGEM distributions connecting different parts functionally for totally new applications. Of course, the output RNA could for example code for a fluorescent protein such as GFP or another GOI. But, since the output is again RNA, it can directly function as input for another switch - and serve as transmitter molecule in the context of a logical circuit.
So the major advantage over conventional protein based devices and even riboswitches is the small size of our basic units, its easy construction, huge variability and easy upscaling to complex networks in cells. Furthermore introduction of an RNA network into E. coli is especially easy - all informations can be coded on one plasmid. In comparison to protein networks, the elegance of RNA based networks lies in its functionality, simplicity and predictability. Since RNA-RNA interactions are highly predictable and since our system is based on the variation of one principle in many switches, simulations of our networks are much more accurate than those of comparable systems currently available. Incorporation of established Biobricks allows a high diversity of input and output signals providing a whole new level of cell control.

Network construction

Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks.
To read more about this, look at our Software page

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Our Objective

Putting the implementation described above into pratise, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to modify a transcription terminator, in such a way, that it interacts with a second RNA molecule and as a result is no longer capable of form a stem loop.
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.

Evaluation and Measurements

To evaluate the functionality of our molecular switches, we first had to establish several assays. Therfore, we imporved and tested an existing in vivo assay as well as developed an in vitro assay. For more information please refer to the lab section.

Results

Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility in vivo, in vitro and in silico. Furthermore we developed a software which allows easy constructions of networks delivering a ready network. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.

in silico design of switching and trigger unit

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attenuation principle

A random sequence was derived from xxx. A complementary sequence, reaching within the terminator´s stem loop was stepwise shortened to find the length, where the formation of the terminator is thermodynamically favored compared to the strand displacement by the signal. The trigger sequence was defined by selecting the shortest unit which still is able to "destroy" the stem loop. Subsequently, the trigger unit was tested in regard of not being able to resolve the stem loop on its on. As table xxx illustrates, the terminator is thermodynamically favorite toward the trigger unit, but in combination with the specificity site, binding becomes possible.


element free energy [kcal/mol] ratio of paaring [%]
switch (terminator secondary structure) trigger unit + switch signal (specificity site + trigger unit) + switch
-30.10 -24.21 -72.88
0 100



Dotplot switch.png
Switch.png
Dotplot triggerunit+switch.png
Signal+switch.png
Dotplot signal+switch.png



















































tiny abortive principle

ubiquitous terminators

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Modeling

To read how bioLOGICS switches theoretically should be able to interrupt termination check out the Modeling page.

in vivo functionality screening

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in vitro screening

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in vitro translation

in vitro transcription

malachite green assay

RNA-PAGE

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Software

To learn how bioLOGCIS theoretically would allow the construction of complex information processing networks check out the Software page.

References

[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi [2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation [3] https://2009.igem.org/Team:TUDelft [4] https://2008.igem.org/Team:Heidelberg [5] Smolke and so on.... [6] http://en.wikipedia.org/wiki/Logic_gate#Symbols [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]


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